Hypothesized life cycle of the snow algae Chlainomonas sp. (Chlamydomonadales, Chlorophyta) from the Cascade Mountains, USA.

Chlainomonas (Chlamydomonadales, Chlorophyta) is one of the four genera of snow algae known to produce annual pink or red blooms in alpine snow. No Chlainomonas species have been successfully cultured in the laboratory, but diverse cell types have been observed from many field-collected samples, from multiple species. The diversity of morphologies suggests these algae have complex life cycles with changes in ploidy. Over 7 years (2017-2023), we observed seasonal blooms dominated by a Chlainomonas species from late spring through the summer months on a snow-on-lake habitat in an alpine basin in the North Cascade Mountains of Washington, USA. The Bagley Lake Chlainomonas is distinct from previously reported species based on morphology and sequence data. We observed a similar collection of cell types observed in other Chlainomonas species, with the addition of swarming biflagellate cells that emerged from sporangia. We present a life cycle hypothesis for this species that links cell morphologies observed in the field to seasonally available habitat. The progression of cell types suggests cells are undergoing both meiosis and fertilization in the life cycle. Since the life cycle is the most fundamental biological feature of an organism, with direct consequences for evolutionary processes, it is critical to understand how snow algal life cycles will influence their responses to changes in their habitat driven by climate warming. For microbial taxa that live in extreme environments and are difficult to culture, temporal field studies, such as we report here, may be key to creating testable hypotheses for life cycles.

Comparative metatranscriptomics identifies molecular bases for the physiological responses of phytoplankton to varying iron availability.

In vast expanses of the oceans, growth of large phytoplankton such as diatoms is limited by iron availability. Diatoms respond almost immediately to the delivery of iron and rapidly compose the majority of phytoplankton biomass. The molecular bases underlying the subsistence of diatoms in iron-poor waters and the plankton community dynamics that follow iron resupply remain largely unknown. Here we use comparative metatranscriptomics to identify changes in gene expression associated with iron-stimulated growth of diatoms and other eukaryotic plankton. A microcosm iron-enrichment experiment using mixed-layer waters from the northeastern Pacific Ocean resulted in increased proportions of diatom transcripts and reduced proportions of transcripts from most other taxa within 98 h after iron addition. Hundreds of diatom genes were differentially expressed in the iron-enriched community compared with the iron-limited community; transcripts of diatom genes required for synthesis of photosynthesis and chlorophyll components, nitrate assimilation and the urea cycle, and synthesis of carbohydrate storage compounds were significantly overrepresented. Transcripts of genes encoding rhodopsins in eukaryotic phytoplankton were significantly underrepresented following iron enrichment, suggesting rhodopsins help cells cope with low-iron conditions. Oceanic diatoms appear to display a distinctive transcriptional response to iron enrichment that allows chemical reduction of available nitrogen and carbon sources along with a continued dependence on iron-free photosynthetic proteins rather than substituting for iron-containing functional equivalents present within their gene repertoire. This ability of diatoms to divert their newly acquired iron toward nitrate assimilation may underlie why diatoms consistently dominate iron enrichments in high-nitrate, low-chlorophyll regions.

Identification of G protein-coupled receptor signaling pathway proteins in marine diatoms using comparative genomics.

BACKGROUND: The G protein-coupled receptor (GPCR) signaling pathway plays an essential role in signal transmission and response to external stimuli in mammalian cells. Protein components of this pathway have been characterized in plants and simpler eukaryotes such as yeast, but their presence and role in unicellular photosynthetic eukaryotes have not been determined. We use a comparative genomics approach using whole genome sequences and gene expression libraries of four diatoms (Pseudo-nitzschia multiseries, Thalassiosira pseudonana, Phaeodactylum tricornutum and Fragilariopsis cylindrus) to search for evidence of GPCR signaling pathway proteins that share sequence conservation to known GPCR pathway proteins. RESULTS: The majority of the core components of GPCR signaling were well conserved in all four diatoms, with protein sequence similarity to GPCRs, human G protein α- and ß-subunits and downstream effectors. There was evidence for the Gγ-subunit and thus a full heterotrimeric G protein only in T. pseudonana. Phylogenetic analysis of putative diatom GPCRs indicated similarity but deep divergence to the class C GPCRs, with branches basal to the GABAB receptor subfamily. The extracellular and intracellular regions of these putative diatom GPCR sequences exhibited large variation in sequence length, and seven of these sequences contained the necessary ligand binding domain for class C GPCR activation. Transcriptional data indicated that a number of the putative GPCR sequences are expressed in diatoms under various stress conditions in culture, and that many of the GPCR-activated signaling proteins, including the G protein, are also expressed. CONCLUSIONS: The presence of sequences in all four diatoms that code for the proteins required for a functional mammalian GPCR pathway highlights the highly conserved nature of this pathway and suggests a complex signaling machinery related to environmental perception and response in these unicellular organisms. The lack of evidence for some GPCR pathway proteins in one or more of the diatoms, such as the Gγ-subunit, may be due to differences in genome completeness and genome coverage for the four diatoms. The high divergence of putative diatom GPCR sequences to known class C GPCRs suggests these sequences may represent another, potentially ancestral, subfamily of class C GPCRs.

Patchy and Pink: Dynamics of a Chlainomonas sp. (Chlamydomonadales, chlorophyta) algal bloom on Bagley Lake, North Cascades, WA.

Snow algal blooms frequently occur throughout alpine and polar environments during spring and summer months; however, our understanding of bloom dynamics is limited. We tracked a recurrent bloom of Chlainomonas sp. on Upper Bagley Lake in the North Cascade Mountains, USA, to assess the spatiotemporal dynamics in bloom color intensity, community photophysiology, and community composition over eight weeks. We found that the algae biomass had a dynamic patchy distribution over space and time, which was decoupled from changes in community composition and life-cycle progress averaged across the bloom. The proportional representation of Chlainomonas sp. remained consistent throughout the study while the overall community composition shows a progression through the bloom. We found that community photophysiology, measured by the maximum quantum yield of PSII (Fv/Fm), decreased on average throughout the bloom. These findings suggest that the Chlainomonas sp. community on Bagley Lake is not simply an algal bloom with rapid increase in biomass followed by a population crash, as is often seen in aquatic systems, though there is a physiological trajectory and sensitivity to environmental stress. These results contribute to our understanding of the biology of Chlainomonas sp. and its response to environmental stress, specifically an extreme warming event.

Large spinose microfossils in Ediacaran rocks as resting stages of early animals.

Large (>100 microm), profusely ornamented microfossils comprise a distinctive paleontological component of sedimentary rocks deposited during the Ediacaran Period (635-542 million years ago). Smaller spinose fossils in Paleozoic rocks have commonly been interpreted as algal cysts or phycomata, but the Ediacaran populations differ from modern algal analogs in size, shape, ultrastructure, and internal contents. In contrast, cysts formed during the diapause egg-resting stages of many metazoans share features of size, ornamentation, and internal contents with large ornamented Ediacaran microfossils (LOEMs). Moreover, transmission electron microscopic observations of animal-resting cysts reveal a 3-layer wall ultrastructure comparable to that of LOEM taxa. Interpretation of these distinctive Ediacaran microfossils as resting stages in early metazoan life cycles offers additional perspectives on their functional morphology and stratigraphic distribution. Based on comparisons with modern marine invertebrates, the recalcitrant life stage represented by LOEMs is interpreted as an evolutionary response to prolonged episodes of bottom water anoxia in Ediacaran shelf and platform environments. As predicted by this hypothesis, the later Ediacaran disappearance of LOEM taxa coincides with geochemical evidence for a marked decline in the extent of oxygen-depleted waters impinging on continental shelves and platforms. Thus, the form, diversity, and stratigraphic range of LOEMs illuminate life cycle evolution in early animals as influenced by the evolving redox state of the oceans.

The earliest history of eukaryotic life: uncovering an evolutionary story through the integration of biological and geological data.

While there is significant data on eukaryogenesis and the early development of the eukaryotic lineage, major uncertainties regarding their origins and evolution remain, including questions of taxonomy, timing, and paleoecology. Here we examine the origin and diversification of the eukaryotes in the Proterozoic Eon as viewed through fossils, organic biomarkers, molecular clocks, phylogenies, and redox proxies. Our interpretation of the integration of these data suggest that eukaryotes were likely aerobic and established in Proterozoic ecosystems. We argue that we must closely examine and integrate both biological and geological evidence and examine points of agreement and contention to gain new insights into the true origin and early evolutionary history of this vastly important group.

Sterols in a unicellular relative of the metazoans.

Molecular clocks suggest that animals originated well before they first appear as macroscopic fossils, but geologic tests of these hypotheses have been elusive. A rare steroid hydrocarbon, 24-isopropylcholestane, has been hypothesized to be a biomarker for sponges or their immediate ancestors because of its relatively high abundance in pre-Ediacaran to Early Cambrian sedimentary rocks and oils. Biolipid precursors of this sterane have been reported to be prominent in several demosponges. Whether 24-isopropylcholestane can be interpreted as a sponge (and, hence, animal) biomarker, and so provide clues about early metazoan history, depends on an understanding of the distribution of sterol biosynthesis among animals and their protistan relatives. Accordingly, we characterized the sterol profile of the choanoflagellate Monosiga brevicollis, a representative of the unicellular sister group of animals. M. brevicollis does not produce a candidate sterol precursor for 24-isopropylcholestane under our experimental growth conditions. It does, however, produce a number of other sterols, and comparative genomics confirms its biosynthetic potential to produce the full suite of compounds recovered. Consistent with the phylogenetic position of choanoflagellates, the sterol profile and biosynthetic pathway of M. brevicollis display characteristics of both fungal and poriferan sterol biosynthesis. This is an example in which genomic and biochemical information have been used together to investigate the taxonomic specificity of a fossil biomarker.

pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree.

BACKGROUND: Likelihood-based phylogenetic inference is generally considered to be the most reliable classification method for unknown sequences. However, traditional likelihood-based phylogenetic methods cannot be applied to large volumes of short reads from next-generation sequencing due to computational complexity issues and lack of phylogenetic signal. "Phylogenetic placement," where a reference tree is fixed and the unknown query sequences are placed onto the tree via a reference alignment, is a way to bring the inferential power offered by likelihood-based approaches to large data sets. RESULTS: This paper introduces pplacer, a software package for phylogenetic placement and subsequent visualization. The algorithm can place twenty thousand short reads on a reference tree of one thousand taxa per hour per processor, has essentially linear time and memory complexity in the number of reference taxa, and is easy to run in parallel. Pplacer features calculation of the posterior probability of a placement on an edge, which is a statistically rigorous way of quantifying uncertainty on an edge-by-edge basis. It also can inform the user of the positional uncertainty for query sequences by calculating expected distance between placement locations, which is crucial in the estimation of uncertainty with a well-sampled reference tree. The software provides visualizations using branch thickness and color to represent number of placements and their uncertainty. A simulation study using reads generated from 631 COG alignments shows a high level of accuracy for phylogenetic placement over a wide range of alignment diversity, and the power of edge uncertainty estimates to measure placement confidence. CONCLUSIONS: Pplacer enables efficient phylogenetic placement and subsequent visualization, making likelihood-based phylogenetics methodology practical for large collections of reads; it is freely available as source code, binaries, and a web service.

Measuring Cluster Stability in a Large Scale Phylogenetic Analysis of Functional Genes in Metagenomes Using pplacer.

Analysis of metagenomic sequence data requires a multi-stage workflow. The results of each intermediate step possess an inherent uncertainty and potentially impact the as-yet-unmeasured statistical significance of downstream analyses. Here, we describe our phylogenetic analysis pipeline which uses the pplacer program to place many shotgun sequences corresponding to a single functional gene onto a fixed phylogenetic tree. We then use the squash clustering method to compare multiple samples with respect to that gene. We approximate the statistical significance of each gene's clustering result by measuring its cluster stability, the consistency of that clustering result when the probabilistic placements made by pplacer are systematically reassigned and then clustered again, as measured by the adjusted Rand Index. We find that among the genes investigated, the majority of analyses are stable, based on the average adjusted Rand Index. We investigated properties of each gene that may explain less stable results. These genes tended to have less convex reference trees, less total reads recruited to the gene, and a greater Expected Distance between Placement Locations as given by pplacer when examined in aggregate. However, for an individual functional gene, these measures alone do not predict cluster stability.